CZ20021986A3 - Embryogenesis and regeneration of plants from microspores - Google Patents

Abstract

A method for embryogenesis and a method for plant regeneration are disclosed. Microspore-containing plant segment from donor plants are harvested and incubated under pre-treatment conditions to maintain microspores at a uninucleate cell cycle G1 phase. Pre-treatment conditions comprise cold water or an aqueous solution of about 0.2 - about 1.0 mol/liter sugar alcohol, for example mannitol. Microspores are isolated from the plant segment and embryogenesis of microspores is induced in induction medium, thereby producing embryos. Green plants may be regenerated from the embryos produced. Arabinogalactan protein, auxin and ovary co-culture may be added to the induction medium to enhance embryogenesis from microspores.

Description

Place microspore drops on filter paper using vacuum

Induction culture of microspores Embryo differentiation

Growth of donor plants

Harvesting and cleaning of parts of plants containing microspores

Pre-induction preparation of ears or donor plants in Petri dishes for microspore isolation (see Fig. · 4)

He washed the microspores with cold mannitol

Regeneration of embryos and ores of double haploid plants

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EMBRYOGENESIS AND REGENERATION OF MICROSPORTS PLANTS

Technical field

The invention relates to methods for producing embryos and methods for regenerating plants from cultured microspores.

BACKGROUND OF THE INVENTION

Double haploid plants can be generated from haploid plants or cells in which the number of chromosomes is doubled to the normal somatic count (2n) by duplication. Methods for producing haploid and doubled haploid plants have utility in plant breeding and transgenic plant production.

Induction of microspore division leading to embryogenesis has utility in the production of large numbers of haploid and doubled haploid plants. Methods are known to induce the formation of embryo-like structures from microspores where genotypes of individual species can be induced to produce a large number of haploids. However, the regeneration of green plants from such embryos under available methodology has proved to be problematic. Genotypes of certain monocotyledonous species, such as cereals, produce albino plants from embryo-like structures. Conventional microspore culture isolation techniques have not yet resulted in consistent embryo regeneration, and disadvantageously a large proportion of albino plants have been formed.

U.S. Pat. U.S. Patent No. 4,840,906 (Hunter) discloses a method of plant regeneration of barley microspores incubated at 25 ° C for 28 days in a sugar-containing medium after prior pretreatment for 28 days in the cold. The method described very different results in the frequency of formation of green plants from microspores, which in some cases ranged from about 30 to about 200 green plants formed from 100 cultivated anthers. Apparently, there is a need for a better method that consistently produces high yields of green plants from isolated microspores.

US Patent 5,445,961 (Genovesi et al.) Describes a method for embryogenesis of microspores using sugar alcohol and cold pretreatment (about 10 ° C), which also requires colchicine, a chromosome doubling agent. This pre-treatment is followed by isolation of microspores and growth on the nutrient medium. According to this method, from 2 to 115 embryoids were generated for every 10 ⁴ incubated microspores. Following the action of the chromosome doubling agent, the transformation of microspores to homozygous transformants does not lead significantly, homozygous transformants are more likely to arise if the transformation takes place before chromosome doubling.

However, no transgenic microspore transformation is described herein.

Haploid unicellular microspore is an attractive target for mutation, selection and transformation. When the transformation is carried out in the G1 phase of the nuclear cycle, genetically homozygous plants are produced which contain transgenes introduced before division. Yao et al. (Genome 1997: 40: 570-581), to transform microspores, act on highly inducible microspores of barley genotype Igri by mannitol, followed by biolistic bombardment. However, these regenerated plants were largely heterozygous for the introduced transgene.

Deac et al. (Theor. Appl. Genet. 1994; 89: 525-533) describes a method of using microspores isolated from barley for particle bombardment. Microspores are rapidly isolated from the cold-treated ears and kept at 24 ° C for 1 hour before transgene bombardment. According to the method described, an average of 82 green plants per 10 ⁵ microspores were produced and only one transgenic plant was produced for every 2.8 x ^{10 6} microspores.

Methodological conditions that contribute to a successful methodology of plant embryogenesis and regeneration include donor plant growth, pretreatment, nutrient media components such as sugar type, nitrogen source and balance, hormones, environmental density and osmolality. However, a process leading to a high frequency of embryogenesis and the successful production of large quantities of green plants has not been described. There is a need to introduce a methodology that utilizes an optimal and synergistic combination of the above mentioned conditions, which leads to high productivity of green plants from microspores. Such an improved method would save labor and be more efficient than conventional methodology, thus reducing production costs.

Arabinogalactan proteins (AGPs) are plant proteoglycans present in different numbers of plant tissues that may have regulatory functions for plant cell reproductive processes. Earlier reports have illustrated the positive effects of arabinogalactan proteins on somatic embryogenesis in Picea abies (Norway spruce) and Daucus carota L. (see, eg, Egertsdotter et al., Physiol Plant 1995; 93: 334-45; and Kreuger et al., Planta 1993; 189: 243-248. Kawaguchi et al. (Plant Journal, 1996; 9 (6): 777-785) discloses a tetrasaccharide with a similar structure to the arabinogalactan protein,

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which accumulates in sows of rice in a stage-specific manner. The effects of AGP on embryogenesis and plant regeneration from microspores were not evaluated.

European Published Patent Application No. 0 455 597 A1 (Sandoz Ltd.) describes a method for stimulating the growth of Daucus carota L. plant cells in an in vitro culture. This publication describes the stimulation of growth by the addition of AGP nutrient medium in an amount of 0.01 to 100 mg / L. However, AGP has not been used to improve the efficacy in inducing embryogenesis of microspores or has been investigated. In addition, no methodology for transforming or determining the stage of cells in culture has been reported.

There is a need for an embryogenesis method that leads to a high success rate of green plant production.

There is also a need for an efficient method of transforming plants with biogenic induction of microspores, which provides effective pretreatment for microspore synchronization and maintains them prior to transformation in the G1 phase of the cell cycle, thereby increasing production of homozygous transformants.

It is an object of the present invention to overcome the disadvantages of the prior art. This object is achieved by a combination of features of the main claims, the subclaims describe a further preferred embodiment of the invention.

SUMMARY OF THE INVENTION

The invention relates to methods for producing embryos and regenerating plants from cultured microspores.

According to the invention, there is provided a method of producing an embryo comprising the steps of: (a) collecting a plant segment containing microspores from a donor plant; (b) incubating the segment under pretreatment conditions to maintain a substantial fraction of the microspores in the mononuclear phase of the G1 cell cycle; (c) isolating the microspores from the segment; and (d) incubating the isolated microspores in the induction medium containing the arabinogalactan protein to induce embryogenesis and thus embryo production.

Further, the invention provides a method of regenerating plants from microspores, comprising the steps of: (a) collecting a plant segment comprising microspores from a donor plant; (b) incubating the segment under pretreatment conditions to maintain a substantial fraction of the microspores in the mononuclear phase of the G1 cell cycle; (c) isolating the microspores from the segment; (d) incubating the isolated microspores in the auxin-containing induction medium to induce embryo production; (e) incubation of embryos in differentiation

medium for producing differentiated embryos; and (f) regenerating plants from differentiated embryos.

The invention is also directed to the production of an embryo prepared as described above and to the production of a plant formed from the embryo.

The invention also relates to a method of introducing a gene of interest into a microspore, which comprises introducing a genetic structure containing a gene of interest into a microspore, which further undergoes the pre-treatment and isolation steps. Methods for introducing the genetic structure into microspores include particle bombardment or Agrobacterium-mediated transformation. Further, the invention is directed to a transgenic microspore prepared in this manner, a transgenic embryo and a transgenic plant produced from both this transgenic microspore and the transgenic embryo.

Advantageously, the method is effective and saves labor when compared to the prior art methods of forming microspore and anthropine cultures. A higher success rate in the production of green plants according to the new method leads to a reduction in costs. Another advantage of this new methodology is the reduction in the number of albinose plants produced.

Preferably, the novel method is effective for different genotypes, indicating that there is no strong genotypic effect. The synergistic combination of factors leads to a versatile method that can be applied to many plant species.

Preferably, removal of the anther wall during microspore isolation allows for greater nutritional availability of microspores during induction, differentiation and regeneration steps. The use of an embryo pad during regeneration allows observation of embryos placed thereon. The use of a pad also promotes growth and facilitates transfer of embryos to other media or applications.

The pretreatment maintains, at the same stage, a large population of haploid microspores that are constructed according to the method of the invention, which provide better targets for plant mutation, selection and transformation. Using the novel method, transgenic plants can be formed with microspores that are pretreated and then proceeded by any conventional transformation method, such as transgene bombardment.

Pretreatment, which maintains microspores at the same stage of the cell cycle, allows the production of large numbers of embryos and regenerated plants from isolated microspores. The combination of cold and sugar alcohol during pre-treatment allows for a much shorter duration of action than when exposed to cold alone. Almost all genotypes respond to this combination of cold and sugar alcohol • · ··· · • ·

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The use of a combination of cold and sugar alcohol during pre-treatment induces not only a large number of embryoids but also tends to synchronize the division of microspore cells, resulting in greater uniformity of the microspore population. The likelihood of nuclear separation during pre-treatment is reduced by the combination of cold and sugar alcohol in pre-treatment. This can be valuable for methodologies dealing with microspore transformation, gene expression expression testing, in vitro selection, or other microspore utilization.

The use of arabinogalactan protein, or a hydroxyproline-rich glycoprotein having glucose-rich side chain hydrocarbons, results in increased embryogenesis yield of microspores from pre-treated plant segments during embryogenesis induction, thereby enhancing embryogenesis efficiency of the method.

This essence of the invention does not describe all the essential features of the invention, but shows that it can be performed using various combinations of the features described.

The invention relates to a method of embryogenesis and a method of regenerating plants from a microspore culture. These methods are described in more detail below with reference to Figures 1 to 5. Examples of embryogenesis and regeneration of cereal plants are described in more detail, but the invention is not limited to cereals.

The following description illustrates, by way of example, a preferred embodiment and is not intended to limit the combinations of characteristics necessary to practice the invention.

By "plant segment containing microspores" is meant any portion of the donor plant that may contain microspores for isolation. Examples of such segments include, but are not limited to, shoots, ears, anthers, flowers, fiber, or panicles.

The term "initial treatment" of plant segments containing microspores refers to processes that are performed after the segment has been removed but before pre-treatment. Initial treatment procedures include alcohol or bleach purification and washing or incubation in water as described below.

By "pretreatment" is meant procedures that are performed on plant segments containing microspores after initial treatment and prior to placement on or in an embryogenic induction medium. Such procedures include cold incubation in water, a high-osmotic pressure sugar alcohol solution, or a combination thereof. The pretreatment creates conditions that serve to stop the microspore cell cycle at the G1 phase of the mononuclear cell cycle, maintain substantially all microspores at this mononuclear stage of development, and trigger the subsequent induction of embryogenesis. The pretreatment prepares mononuclear microspores for embryogenic induction by maintaining a synchronized microspore population having from about 50% to 100% microspores to a consistent G1 phase of the cell cycle.

There are three stages that independently control the production of haploid plants from microspores, namely the induction of embryogenesis; regeneration of embryo-like structures; and developing embryo-like structures on green plants. To complete an effective product, it is necessary to optimize the conditions of each stage.

For efficient production of green homozygous plants from a microspore culture, a number of factors need to be controlled. Each factor affects one of the above stages in the production of haploid plants from microspores. In order to implement a successful method of growing homozygous plants from a microspore culture, various factors such as donor plant growth conditions, ear collection, microspore stage determination, pretreatment, microspore isolation, induction conditions, differentiation conditions, embryo withdrawal conditions, and plant regeneration conditions need to be optimized.

According to the invention, as well as illustrated in the flowchart of Fig. 1, there is provided a method of producing an embryo comprising the steps of: (a) collecting a plant segment containing microspores from a donor plant; (b) incubating the segment under pretreatment conditions to maintain a substantial fraction of the microspores in the mononuclear phase of the G1 cell cycle; (c) isolating the microspores from the segment; and (d) incubating said isolated microspores in an incubation medium comprising arabinogalactan protein and amino acids to induce embryogenesis and thus embryo production.

The invention further relates to a method for regenerating a plant from microspores, comprising the steps of: (a) collecting a plant segment comprising microspores from a donor plant; (b) incubating the segment under pretreatment conditions to maintain a substantial fraction of the microspores in the mononuclear phase of the G1 cell cycle; (c) isolating the microspores from the segment; (d) incubating the isolated microspores in an induction medium containing auxin, high levels of glutamine and amino acids to produce embryos; (e) incubating the embryos in the differentiation medium to produce differentiated embryos; and (f) regenerating plants from differentiated embryos.

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Plant growth

According to the invention, it is desirable for the donor plants to be grown in a controlled environment which is preferably free of pests, but the invention is not directed only to plants grown under these conditions. The following growth conditions in a controlled environment are given by way of example and are not intended to be limiting in any way.

The optimum relative humidity may range from about 50% to about 90%, preferably from about 60% to about 80%. The illumination ranges from about 300 pEm ² s to about 450 pEm ² s, preferably from about 350 pEm ² s to about 400 pEm ² s at the pot level. Any suitable light source such as a Gro-Lux ™ white fluorescent lamp, incandescent lamp, or a combination thereof can be used. Plants can be grown in nutrient solutions.

More consistent yields of embryos and green plants arise from microspores produced in plants that have not suffered from drought, stress or pest infestation, or combinations thereof. Plants exposed to stress and produced either under inadequate or excessive irrigation, or under attack by pests, often produce microspores that react inconsistently or their culture may die.

The following temperatures lead to optimal plant growth. For winter barley, these are temperatures from about 13 to about 17 ° C, preferably from about 15 ° C in the light for about 15 to about 18 hours, preferably from about 16 to about 17 hours per day. During a dark cycle that lasts from about 6 to about 9 hours, preferably from about 7 to 8 hours per day, optimal temperatures are from about 10 to about 14 ° C, preferably about 12 ° C.

For spring barley that grows in a light cycle that lasts 15-18 hours, preferably 16-17 hours, temperatures can range from 16 to 20 ° C, preferably 18 ° C.

During a dark cycle that lasts 6-9 hours, preferably 7-8 hours per day, the optimal temperatures for spring barley are from about 13 to 17 ° C, preferably 15 ° C.

For winter or spring hexaploid wheats that grow in a light cycle lasting 15-18 hours, preferably 16-17 hours, temperatures can range from 16 to 25 ° C. During a dark cycle of 6-9 hours, preferably 7-8 hours per day, optimal temperatures for hexaploid wheat range from 13 to 18 ° C, preferably 15 to 16 ° C.

Plants can be fertilized once a week to optimize growth. Spraying with pesticides during the harvesting period should be avoided as spraying could reduce the microspore response. However, plants can be sprayed at an earlier stage to minimize pest or fungal attack.

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Determination of microspore stage

Plant segments containing microspores, such as shoots, ears, panicles, anthers, flowers or fiber, are collected from donor plants when the oldest microspores are in the middle or late mononuclear stage.

Optionally, the microspore sample from the plant segment containing the microspores may be inspected to ensure a suitable stage of development. A description of the mid to late mononuclear stage can be found in publications such as Wheatly et al. (Plant Cell Rep 1986; 5; 47-49), the disclosure of which is incorporated herein by reference. One of the most prominent features of this stage is the location of the core relative to the vent (see Figure 2). In the initial mononuclear stage, the nucleus is near the vent, and in the late mononuclear stage it is opposite the vent. To illustrate this phenomenon, barley microspore pickling with acteocarmine or DAPI can be used. Alexander's stain can be used to illustrate this phenomenon in microspores derived from wheat and corn.

In the case where the plant segment containing the microspores is a shoot, the ears are removed from the sheath of the shoots. Microspores obtained from the flower located in the central part of the ears can be examined by determining the stage of the cells to determine the stage of development. Subsequently, plant segments containing microspores in which the microspores are near the mid to late mononuclear stage may be selected based on their developmental similarity to those investigated by pickling.

Alternatively, the ears can be removed from the vagina by comparing the consistent length and appearance of the flowers to the segment of interest.

Initial treatment of plant segment containing microspores

Selected plant segments containing microspores having microspores in the middle or late mononuclear stage are initially placed in petri dishes (e.g., 15x100 mm) prior to pretreatment, which is described in more detail below.

Optionally, plant segments containing microspores can be cleaned with a cleaning solution. Suitable cleaning solutions may include ethanol solutions containing from about 50% to 80% ethanol, preferably about 70% ethanol, or aqueous solutions containing about 10% to about 15% bleach (about 0.5% to about 1.0% chlorine) .

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For example, and is not intended to be limiting in any way, the separated shoots may be sprayed with an aqueous solution of about 70% ethanol prior to removal of the ears. In barley, the pains can then break off and the ears undergo pre-treatment as described in more detail below. For wheat, if it has pains, the pains can be removed, but not too close, as exposing the flower to the inside of the cleaning solution could be harmful.

Another example might be the case, which should not be considered in any way limiting, when pain or ears grow out of the leaf sheath or if there is a problem with contamination, ears can be sterilized by placing them in a glass jar and pouring for 10-25 minutes solution of about 10% bleach. Isolated anthers are not treated with bleach. After cleaning, the flowers and ears are washed well with water and the residual moisture is allowed to evaporate before incubation in mannitol solution. Cleaning can be carried out in Flow Bench to ensure sufficiently clean solutions.

As a further option, during initial treatment, selected microspore-containing plant segments may be subjected to post-harvest incubation in ice water at a temperature of about 3 ° C to about 6 ° C, preferably about 4 ° C for up to 4 weeks, e.g. for 3 to 4 weeks. For example, ears may be incubated in sterile water in petri dishes (about 0.5 ml water per dish) at about 4 ° C for about 2-4 weeks. This incubation can be performed if, for example, the segment is harvested before the microspores are at a suitable stage of development. Therefore, the post-incubation incubation can continue until the segment reaches a suitable stage of the microspore to allow pre-treatment as determined by pickling. For example, the shoots can be incubated in ice water until the ears have been removed from the vagina. The post-incubation incubation period should be minimized to prevent microspore stage progression during this period.

Before editing

Pretreatment of plant segments containing microspores is performed to unify the microspores to a uniform stage of development in preparation for embryogenic induction. The pretreatment comprises the use of cold in combination with incubation of plant segments in sterile water or a sterile aqueous sugar alcohol solution. For some species, this pretreatment may require the addition of medium with macrosols.

The pretreatment may take from about 3 days to about 4 weeks, depending on the conditions used. If water is used during cold incubation, duration

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The pretreatment may vary from 1 week to about 4 weeks depending on the species and genotype. When a sugar alcohol solution is used, a shorter incubation period of from about 3 to about 5 days may be used, where 3 days are preferred for transformation procedures.

For pretreatment in a sugar solution, the sugar alcohol concentration may range from 0.1 mol / l to about 1.0 mol / l. Preferably, the sugar alcohol is present in an amount from about 0.2 mol / l to about 0.5 mol / l, and more preferably is in an amount of about 0.3 mol / l for barley, or 0.4 mol / l for wheat. The sugar alcohol may be selected from any alcohol contained in the group: mannitol, maltitol, sorbitol, xylitol, or any combination thereof, and preferably mannitol may be used. Treatment of ears, where the preferred combination is cold (4 ° C) with incubation in mannitol (0.3 mol / l) for 3 days, increases the yield of green plants regenerated from microspores.

The sugar alcohol is mixed with the plant segments in an adequate amount to partially overlap the segments that are stored in a single layer, for example about 15 ml in a 15x100 mm petri dish. Ph treatment with ears of ice water, only 0.5 ml of water is added to a 15 x 100 mm dish.

The petri dishes are then sealed, for example, with a film such as Parafilm ™ and placed in the dark at a temperature of from about 3 ° C to about 6 ° C, preferably at about 4 ° C. Incubation in the sugar alcohol solution is continued for about 3 to 5 days and incubated in ice water for about 10 days to about 4 weeks, depending on the species.

Alternatively, plant segments may be incubated at room temperature according to the method of Roberts-Oehlschlger and Dunwell (1990) (about 22 ° C to about 27 ° C, preferably at about 25 ° C) in an aqueous solution containing from about 0.2 moles. to about 0.5 mol / l sugar alcohol, preferably about 0.3 mol / l sugar alcohol. This incubation for up to 4 days, preferably for 3-4 days, effectively triggers induction, but does not maintain microspores on the mononuclear phase.

Microspore insulation

After pre-treatment, the microspores are isolated from plant segments. If the plant segments are anthers, they can be processed in the vortex chamber for up to 10 minutes, preferably about 5 minutes, when most microspores are separated as previously described by Kasha et al. (IAEA-SM-1995: 340/9: 23-37).

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Glass and tools for collecting plant segments after pre-treatment should be sterilized (autoclaved) and Flow Bench. The ear or fiber segments with flowers are removed to reduce the segment size to a length of about 2 cm to about 3 cm. The cut segments are then poured over with an ice-cold aqueous sugar alcohol solution containing from about 0.2 mol / l to about 0.5 mol / l sugar alcohol, preferably from about 0.3 mol / l mannitol to form a suspension. The suspension is stirred at a rate suitable to release microspores from the rest of the plant segment.

When stirring the slurry, a cooled mixer is preferably used. An adequate amount of chilled sugar alcohol solution is added to the plant segments to cover the ear segments. By way of illustration, to obtain a sufficient amount of microspores (500,000) per dish, eight to ten barley ears may be poured in two rows in a mixer, or six rows of barley or wheat ears of about 200-300 ml of chilled sugar alcohol solution. During mixing, the plant segments and the sugar alcohol solution are mixed at low speed for up to 15 seconds, preferably from about 5 to 10 seconds. Nevertheless, the appropriate mixing time and speed vary depending on genotype and pretreatment, and ultimately affects viability.

As an alternative to mixing with limited availability of ear counts, an insulating process in the swirl chamber can be applied to insulated anthers. This procedure improves the viability of the microspores since the vortex chamber insulation is more gentle to the segments to be treated than mixing (see, e.g., Kasha et al. IAEA-SM-1995: 340/9: 23-37).

The resulting microspore-containing slurry is quickly filtered and washed with a sugar alcohol solution (see FIG. 3), for example at a concentration of 0.2 mol / l to about 0.5 mol / l, preferably 0.3 mol / l mannitol. Care should be taken to avoid plasmolysis of microspores, as embryos would not develop from plasmolyzed microspores. If the microspore lifetime is below 50%, dead microspores can be removed by placing the resuspended tablet in a density gradient, such as a 20% maltose density gradient followed by centrifugation, and retaining a viable microspore fraction.

Embryogenic induction of isolated microspores

After pretreatment and isolation, microspores are induced to develop into embryos. After the final wash in the microspore isolation step, the supernatant is decanted and the cake is decanted. * »· * # # #

4. with microspores resuspend in induction medium. The induction medium optimizes the plant's response to induction of embryogenesis.

The induction medium may comprise any plant culture medium containing suitable macro- and microsols to support viable microspores to which auxin has been added. Examples of such media for use as induction media, which is in no way intended to be limiting, are shown in Table 1. These media are based on FHG media as described by Hunter (1987) and MS media as described by Murashige and Skoog (1962). ). These basal media were modified for use in barley (MFHG) and wheat (MMS4).

One possible auxin that can be added to the basic medium of the invention is PAA (phenylacetic acid), which is effective in providing an induction response (Ziauddin et al. 1990). In the composition of Table 1 exemplified and in no way intended to be limiting, the induction medium contains a high amount of glutamine, maltose as a sugar and contains PAA as an auxin.

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Table 1: Nutrient composition of MFHG base MMS4

Makrosoli KNOj 1900 1900 1900 1400 NH4NO3 165 165 1650 300 KHjPO ₄ 170 170 170 170 MgSO ₄ 7H, O 370 370 370 370 CaCl, -2H, O 440 440 440 440 FeSO ₄ .7H2O - - 27.8 27.8 Mikrosoli j FeNayEDTA 40 40 37.3 37.3 MnSO ₄ -5H ₃ O 22.3 22.3 22.3 22.3 H ₃ BOj 6.2 6.2 6.3 6.3 ZnSO ₄ -7H ₂ O 8.6 8.6 8.6 8.6 CoCl 3 -? H? O 0.025 0.025 0.025 0.025 CuSO ₄ -5 H ₂ O 0.025 0.025 0.025 0.025 NaMoO ₄ -2H, O 0.25 0.25 0.25 0.25 KI ♦ - 0.83 0.83 Other components glutamine 730 750 146 975 inositol 100 ALIGN! 100 ALIGN! 100 ALIGN! 300 thiamine 0.4 0.4 0.4 0.4 nicotinic acid - - 0.5 0.5 pyroxidine - - 0.5 0.5 sucrose - - 30.0 gm - maltose 62 gm 62 gm - 90 gm phenylacetic acid - 10 - 4 indole acetic acid - - 1.0 - BAP 1.0 1.0 - - kinetin - - 1.0 0.5 AGP - 10 - 10 agarose - - 8.0gm - fytagel - 3.0gm - 3.0gm

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Modified FHG medium (MFHG) can be used to induce barley isolated microspores, while MMS4 can be used for wheat. Once embryos show signs of green, both barley and wheat are usually regenerated in the original MS medium, except when the kinetin is reduced, for example (but not limited to), to about 30 mg / l. In the intermediate step of embryo differentiation prior to regeneration, suitable induction media can be modified in barley and wheat by reducing maltose, for example (but not limited to), to about 30 mg / l, removing auxin, altering inositol concentration, for example (but not limited to) 250 mg / l, adding casein for example (but not limited to) to a concentration of about 1.0 mg / l and adding proline for example (but not limited to) to a concentration of about 690 mg / l.

The induction media comprises an isotonic combination of salts, an amide such as (but not limited to) glutamine in an amount of about 500 to about 1000 mg / l, a sugar source such as maltose, sucrose, or melobiose, or other sugars (see Hunter, 1988), in an amount of from about 20 g / l to about 100 g / l. Auxin may be used in an amount of from about 2 mg / L to about 20 mg / L, preferably from about 2 mg / L to about 10 mg / L. As auxin, PAA (phenylacetic acid), NAA, picloram (highly potent herbicide), or 2,4-D may be selected (but not limited to). 2,4-D is a stronger auxin and a lower amount is sufficient. An example of such an amount of PAA in the induction medium is from about 4 mg / L to about 5 mg / L. When 2,4-D is used, it is optimal to remove and replace with PAA after 1-4 hours to obtain a high embryo formation rate.

The microspores are initially incubated in an induction medium at a density that provides adequate separation of the microspores. By way of example, and not by way of limitation, incubation of a cake containing about 500,000 barley microspores on sterile and washed filter paper. As another example, 100,000 mixed-crop wheat microspores with seedlings on sterile and washed filter paper.

To enhance the response of microspores in the induction medium, an arabinogalactan protein, referred to herein as AGP, or similar high-galactose-rich hydrocarbon side chain glycoproteins that are rich in hydroxyproline (GaRGP) may be added to the induction medium, which addition improves the microspore reaction. those microspores that are isolated from cereals, such as wheat or barley. The presence of AGP or GaRGP in the induction medium improves the viability of the micro-spores in culture, both in the presence and absence of testes in the mixed culture. The AGP or GaRGP may be added to the induction medium in an amount of from about 1 mg / L to about 100 mg / L of the induction medium, and preferably in the range of about 10 mg / L to about 25 mg / L. In the presence of AGP or GaRGP, the incubation period is optimal for induction from about 10 to about 14 days.

The pH of the induction medium is adjusted in the range of about 5.6 to 6.0, preferably about 5.8. Optionally, the induction medium can be solidified by adding about 3 g / L of a solidifying agent, such as Phytagel ™ or Gelrite ™, to the base liquid medium. When Gelrite ™ is used, higher levels of antibiotics or herbicides are required to achieve effective selection compared to Phytagel ™.

For example, and in no way to be considered limiting, a sterilized Whatman # 2 filter paper is placed on the vacuum area of the vacuum filter flask. Preferably, the support is purified with a liquid medium such as FHG medium, which may or may not contain auxin, an aqueous sugar alcohol solution (about 0.2 mol / l to about 0.5 mol / l, preferably about 0.3 mol / l) ), or sterile distilled water (about 10 to 20 ml). At low vacuum, about 1 ml is pipetted onto the support dropwise into about 2 ml of microspore medium, which may or may not contain auxin.

In total, from about 500,000 to about 600,000 barley microspore embryos can be placed on each support. For wheat microspores, the optimum lower embryo density is from about 50,000 to about 200,000 microspores per support. One skilled in the art can readily determine the optimum microspore density for a given cereal variety for a single substrate. The pad must contain a suitable pore size to allow the passage of liquid, leaving microspore embryos on the pad surface.

At this stage, it is preferred to add a mixed testicle culture to the induction medium in wheat. If the testicles are located between the microspores in the induction medium, embryo development will improve. The seedlings can be isolated from the ears when the microspores are in the mononuclear stage and if they are stored in a cool filter paper until they are used in a mixed culture with isolated microspores. From about 4 to about 20 seedlings per dish may be used. In the presence of AGP, 4 to 6 testicles are sufficient for good microspore response. The seedlings are placed between the microspores.

The microspore pad, and optionally the wheat seedling on the surface, is transferred to trays containing a hardened regeneration medium such as modified FHG (MFHG) (for barley) or MS (MMS4) (for wheat) media described.

9 · · 9 9 · · 9 9 · ί ϊ. J 9 9 99 99 999999 in Table 1 to which regeneration medium components have been added. The dishes are sealed with a gas-permeable foil such as Parafilm ™. More than two layers of Parafilm could cause culture response failure, probably due to insufficient gas exchange. Alternatively, the small, closed dish together with the small, open dish containing the drops of distilled water may be overlaid with a larger dish. In this embodiment, only the large outer bowl is sealed.

To induce embryos, sealed dishes are placed in the dark at temperatures from about 22 ° C to about 30 ° C, preferably about 26 ° C for barley and about 28 ° C for wheat, for about 2 to about 5 weeks. If the filter paper mat dries, droplets of liquid induction medium can be added to the trays.

The microspores are placed on the hardened induction medium and the embryos are allowed to grow to a size of about 1 to about 2 mm, which may take up to four weeks. Smaller embryonic structures may remain on the induction medium dish and one or two drops of freshly prepared induction medium are added to the dish. These structures can be displaced once they have reached the appropriate size. The barley microspores may optionally be cultured in either liquid or agarose-hardened FHG (MFGH) medium or, in wheat, in MS (MMS4) liquid or hardened medium.

Differentiation

Once the embryos reach a size of about 1 mm to about 2 mm, they are transferred from the induction medium to the differentiation medium for one or two weeks until shoots and roots develop. The differentiating medium contains isotonic salts and may contain sucrose up to 50 g / l. One possible differentiation medium is described in Table 1 and consists of MMS4 medium which additionally contains about 30 g maltose, about 250 mg inositol, about 1000 mg casein and about 690 mg proline, in addition to the original MS medium containing thiamine, pyridoxine, acid nicotinic and BAP. No auxin is present in the differentiation medium. The differentiation medium is adjusted to a pH of from about 5.6 to about 6.0, preferably about 5.8. The medium is then solidified by the addition of a reinforcing agent such as Phytagel ™ or Gelrite ™ in amounts of 3 g / L.

Reinforced auxin-free FHG medium can also be used as differentiation medium. Due to the low glutamine content, the original MS medium is most preferably used in culture as a differentiation medium using Basta herbicide-resistant selection, which is advantageous for differentiation because high glutamine levels reduce the efficiency of the selection.

0 0 0 00 9 0 00 9 0

10 · · 0 ···

Regeneration

The differentiated embryos are transferred from the differentiation medium to the regeneration medium to regenerate the green doubled haploid plants.

The differentiated embryos are placed in a Gelrite-reinforced differentiation medium, such as an MS medium modified as shown in Table 1.

Once the shoots appear, the embryos are optionally transferred to a MS medium-based regeneration medium as shown in Table 1. Embryos are left in the dark for about 3 to about 4 days and then left in moderate light for about 8 hours. day at a temperature of from about 20 ° C to about 25 ° C, preferably about 22 ° C. Small plants (about 3 cm to about 4 cm high) are transferred to larger containers or beakers containing a hormone-free MS regeneration medium to grow the plants. Once the plant has about 3 to 4 leaves, it is placed in strong light and conditioned for about 2 to 5 days. After about 2 to 5 days, the small plants are transferred to soil or pots with growth medium.

Transformation

Transformation of the plants with the gene of interest can be completed using the method described herein by introducing a transgene into the microspores after pretreatment. Since pretreatment shifts microspores to the cell cycle stage prior to nuclear division, the addition of the transgene at this stage allows for further duplication and division of the transgene, resulting in doubled haploid plants that are homozygous for the transgene. Any detectable marker attached to the transgene, such as a herbicide resistance gene such as Bar (Jahne et al. 1994; Yao et al. 1997) or an antibiotic resistance gene, may be suitable for transformation testing. Alternatively, a visible marker such as green fluorescent protein (GFP) can be used (Carlson 1998).

Microspore transformations can be performed using established methods, including particle bombardment (eg Jahne et al., 1994; Yao et al. 1997; Carlson 1998; Yao and Kasha 1998; all of which are incorporated herein by reference) or Agrobacterium transformation (e.g. Or EP 0 737 738, which are incorporated herein by reference). Pre-treated, freshly isolated microspores are preferably used for transformation. An increased frequency of transformation was observed using these described methods. Using the pre-treatment process described herein, combining cold and sugar alcohol, the microspores are maintained on the mononuclear phase of G1, thereby allowing homozygous transgenic plants to be formed.

· · · Φ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · :. ·

0 0 0 0 0

ΦΦΦΦΦΦ Φ ΦΦ ΦΦ ΦΦΦΦΦΦ

By the term "gene of our interest" is meant any gene to be transcribed into the host organism. Such genes of interest include, but are not limited to, a gene whose product has an effect on plant growth or fertility, for example, a plant growth regulator such as auxin or cytokinin and analogs thereof, or a gene of interest may be herbicidal or pesticidal resistance genes, which are well known in the art. Genes of interest may also include a gene that encodes a pharmaceutically active protein, for example, growth factors, growth regulators, antibodies, antigens, derivatives thereof useful in immunization or vaccination, and the like. Examples of such proteins include, but are not limited to, interleukins, insulin, G-CSF, GM-CSF, hPG-CSF, M-CSF or combinations thereof, interferons such as interferon-α, interferon-β, interferon-τ, blood clotting factors , for example Factor VIII, Factor IX, or tPA or combinations thereof. The gene of interest may also encode an industrially produced enzyme, protein dietary supplement, nutritional substance, or feed additive for food, food, or both for feed and food use. Examples of such proteins include, but are not limited to, proteases, oxidases, phytases, chitinases, invertases, lipases, cellulases, xylanases, enzymes involved in oil biosynthesis, etc.

Methods for regenerating whole plants from embryos are also well known in the art. Generally, the transformed microspores are cultured in a suitable medium as described herein, which may contain selective means, such as antibiotics, where selective markers are used to facilitate identification of the transformed microspores that form transgenic embryos. The transgenic embryos then grow into transgenic plants.

The foregoing description is in no way intended to limit the claimed invention, moreover, the combination of characteristics discussed herein may not be necessary for the inventive solution at all.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will be better understood by reference to the accompanying drawings, in which:

Giant. 1 is a workflow diagram representing a method according to an embodiment of the invention.

Giant. 2 illustrates the developmental stages of mononuclear microspores for determining the stage of development of a set of plant segments. In the pictures below are DAPI-stained to:; . ·.::. ·· ..; Microspores in the intermediate or late mononuclear stage where the material is collected as described herein.

Giant. 3 shows the isolation and sowing of microspores. Giant. 3 (a) shows microspore filtration through a canvas. Giant. 3 (b) shows the microspore pellet in a cuvette after centrifugation.

Giant. 3 (c) shows the placement of microspores on filter paper after vacuum filtration.

Giant. 4 illustrates the development of barley microspore embryos c.v. Igri. on filter paper in the absence of AGP. Giant. 4 (a) shows embryos at 3 weeks post-cultures on filter paper. Giant. 4 (b) shows embryos at the age of 5 weeks after introduction of the cultures onto filter paper.

Giant. 5 shows the development of wheat and barley embryos. Giant. 5 (a) shows the development of wheat embryos c.v. Bob White at 4 weeks of age with (bowls on the left) and without (bowls on the right) mixed culture with testes, and further in the presence (upper bowls;

mg / l) and in the absence (bottom plate) of AGP. Giant. 5 (b) shows the development of a barley embryo in the presence of 10 mg / l AGP (left side dish) and in the absence of AGP (right side dish).

Giant. 6 illustrates the proper development of a wheat embryo after 4 weeks in AGP and testicular mixed culture. Giant. 6 (a) shows the development of wheat embryo in culture.

Giant. 6 (b) shows isolated embryos.

The following are examples which describe particular embodiments of the invention. The examples are not to be construed as limiting the invention, but rather, in the exemplary embodiments, those modifications are presented that the person skilled in the art is most familiar with.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

The following procedure is used to isolate the microspores from the slurry obtained by mixing or processing the plant segments in a vortex chamber. Filter the slurry through a canvas or coarse nylon membrane (Fig. 3A). The filtrate is collected and can be filtered again through a nylon mesh, such as 100 µιτι, into a centrifuge tube. The centrifuge cuvette is then centrifuged under appropriate conditions for the formation of the cake from the microspores (Fig. 3B), for example, which does not have the same properties.

4 4 4 4 4 4 4 4 4 9

4 4 4 4 44 ·

4 4 4 9 4 4

4 4 4 4 4 4 4 4 4 in no way intended to limit the invention at 900 rpm. for 5 min. About 0.15 ml of cake can produce approximately 500,000 microspores of barley. The microspore cake is resuspended in a cold aqueous mannitol solution (about 0.2 mol / l to about 0.5 mol / l, preferably 0.3 mol / l mannitol) and washed until the supernatant has a green shade. For example, 2-3 washes can be performed for this purpose. The cake is then suspended in the induction medium and filtered dropwise through filter paper using vacuum filtration (Fig. 3C).

Example 2

Embryogenesis was performed for thirty different barley genotypes according to the procedure described herein. Usually between 10,000 to 15,000 embryos per dish were produced. Table 2 illustrates some embryo data depending on averaging of the three replicates (plates). Values for all thirty genotypes were not counted. However, the response of each genotype was assessed based on the appearance of the plates after two weeks and again after 4-5 weeks. Random samples of 500 embryos were collected from the pans and placed on regeneration medium to determine embryo recovery ability. As shown in Table 2, the number of regenerates ranged from 36 to 97%. Giant. 4 illustrates the development of barley embryo c.v. Igri at 3 weeks of age (Fig. 4 (a)) and 5 weeks (Fig. 4 (b)) on filter paper placed on the hardened induction medium.

• »··« ·

Table 2: Induction and regeneration of embryos after pretreatment of various barley genotypes. · · · · · · · · · · · · · · · · · · · ······

Genotype Before editing Embroids * % of regenerated embroidery Igri 28d in cold, 13136 85% 4 d 0.3M. Man. 4’C 12380 97% 4d 0.3Mman. 25‘C 13504 88% GBC 782 3d 0.3M man. 4‘C 14496 on. GBC 783 3d 0.3M man. 4‘C 11360 on. Harrington 3d 0.3M man. 4’C 5533 36% Lina 21d in cold, 15377 77% 7d 0.2M man 4‘C 8921 81% Oxbow 21 d 0.3M man. 4‘C 10810 58% Manley 4d 0.3M man. 4‘C 11360 74% GBC 777 4d 0.3M man. 4‘C 14133 89% GBC 778 4d 0.3M man. 4’C 7667 84%

28d = 28 days in cold; 0,3M man. 4 ° C = 0.3 mol / L mannitol at 4 ° C; on. = variety not analyzed * The number of embryos is averaged from three dishes, while% regeneration is based on shoots produced on 500 embryos per dish.

The barley genotypes Igri, GBC777 and GBC778 are winter habits, while the rest of the genotypes in Table 2 are spring habits. Harrington, Manley and Oxbow are nowadays the main cultivated two-row malting barley in Canada. Igri and Lina are of European origin. The lines GBC 777 and 778 are six-row barley.

Three different pretreatments on the Igri genotype were compared and no apparent differences between embryo induction and regeneration were observed (Table 2). Lina microspore culture usually responds well to mannitol pretreatment, but the combination of cold with mannitol for 7 days produced only a weak response in this study. Therefore, embryo formation for the Lina variety was calculated for cold pre-treatment for 21 days, when the reaction was more typical of the Lina variety (Table 2). Response to regeneration at »9 · ** ·« 9 ·· 99 99

9 9 9 9 9 9 9 9 9 9. · · ** · · · ·;; ♦ *

9 9 9 9 9 9 9 9 9 * 99 <· ·· 9999 both pre-treatments were similar for Lina. The frequency of fertile doubled haploids in the offspring of Lina after two pretreatments was not different, namely 79% for the combined 7-day pretreatment and 83% after the pretreatment at 21 days in the cold.

Harrington's response was also low for both embryo formation and overall percent recovery, but then improved. Oxbow's response showed that it had a good response at shorter pre-treatment times, but the induced structures had largely become callus. However, if a longer pretreatment is applied, as shown in Table 2, the response is predominantly embryogenic and an acceptable overall recovery rate is obtained. This response is typical of Oxbow, probably due to a higher endogenous auxin content.

The genotype of the 6-line winter habit GBC777 is unusual for producing only about 5% of the green plants and the rest being albino. Even at such a low frequency, it would be possible to receive about 500 green plants per dish if all the embryos were regenerated. Although this procedure would be laborious, it would be possible to transfer all embryos to the culture suspension in the regeneration medium and regenerate the green germinating embryos under slight illumination.

Embryogenesis was visually evaluated in barley winter genotypes:

OAC Elmira, GBC776, GBC779, GBC780 and GBC781, and in spring genotypes, Trinity, Cooper, Golden Promise, Disa, Morex, Excel, Elrose, Bruce, Rodeo, H936 and Sabarilis. Effective embryogenesis was achieved by cold pretreatment with mannitol for 3-4 days in 0.3 mol / l mannitol at 4 ° C for these genotypes. Under these conditions, about 2,000 plants per 10 ⁵ microspores were measured for responsive genotypes, or about 200 plants per ear.

The microspore embryogenesis method of the invention is effective for barley in a wide variety of genotypes. In the above-mentioned barley genotypes, production of embryo structures led to a high percentage of initiation of regeneration.

Example 3

This example discusses the effect of pretreatment time on embryogenesis in barley genotype Igri for ears incubated in 0.3 mol / l mannitol at 4 ° C. Table 3 illustrates that as short a time as 4 days pretreatment drastically affects the number of embryos formed from microspores. Although the number of embryos produced increases up to 28 days of pretreatment, only 4 days of pretreatment are sufficient for beneficial effects. It was found that «·· ·

A r r *> * · A 1 1 A ·

AA * A * A * 9991 pre-treatment time for Igri is not critical to achieving an effective response . However, the pre-treatment time affects the Oxbow variety as previously mentioned and illustrated in Table 2.

Table 3: Effect of pretreatment time on spike embryo formation at 4 ° C in 0.3 mol / l mannitol

Pre-treatment time in days number of embryos produced 0 0 4 9579 7 9693 14 8981 21 9692 28 10213

Example 4

The influence of the use of the nutrient pad on the development of embryos on the regeneration of plants from wheat microspores of the genotype Chris was investigated. Microspores were incubated in induction medium (MMS4 medium) either on a support (Watman # 2 filter paper) or in liquid medium (droplet method). The preparation and use of filter paper has been described above. The droplet method is prepared in the same manner, only the required number of microspores are contained in a concentrated drop of liquid medium which is placed directly on the surface of the solid medium. After 10 days to two weeks, one or two drops of fresh liquid medium are added to both the filter paper and the droplet system to better spread the developing microspores and thus have better development conditions. Table 4 illustrates the results obtained from the three replications. Examples of development are shown in Fig. 4.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··

Table 4: Effect of nutrient pad on embryo development and plant regeneration from wheat microspores (Chris genotypes) induced by MMS4 (without AGP).

Adjustment number bowls number of embryos in the droplet system (ELS) green plants albino plants total % regenerates % of green regenerates filtrační paper 1 650 350 4 354 54 54 2 615 265 3 268 44 43 3 658 390 5 395 60 59 total 1923 1005 12 1017 53 52 drip way 1 300 130 2 132 44 43 2 300 100 ALIGN! 100 ALIGN! 33 33 3 300 110 3 113 38 37 total 900 340 5 345 38.3 37.7

Twice more embryos per plate were induced using a pad during induction than when incubated in liquid induction medium. The number of green plants recovered more than doubled using the pad during induction, further resulting in a higher percentage of regenerated embryo plants. Better development on filter paper may be due to better aeration at the surface of the filter paper. Pretreatment, microspore isolation and medium were the same for both induction and regeneration.

These results demonstrate the benefit of using the mat during embryogenic induction of microspores. Examples of development are shown in Fig. 4.

Example 5

The effect of the regeneration medium composition for wheat microspores that was induced in the medium in the presence and absence of AGP was compared. The regeneration medium was based on MMS4 medium containing either 3% sucrose or 9% maltose as a sugar source, or based on MMS4 medium with 9% maltose and AGP content. Microspores were induced either on a support, in this case on Watman # 2 filter paper, or placed on solid medium with drops.

• · · · · · · · · · · φ · φ · φ · · · · · · · · ·

Table 5: Effect of regeneration medium on the development of plants from wheat microspores (c.a. Pavon 76) induced on AGP medium, either on filters (A) or as drops on solid medium (B), 4 replicates (dishes) were performed.

Regeneration medium # ELS (= number of embryos per drop system) number green plant number albino plant % regenerates % of green regenerates AND MMS ₄ (S *) 498 385 10 79.3 77.3 MMS ₄ (M *) 498 235 22nd 51.6 47.2 AGP (M) 498 148 17 33.1 29.7 Total 1494 768 49 54.7 51.4 May transmission** 600 291 17 51.3 48.5 MMS ₄ (S *) 260 172 6 68.5 66.2 MMS ₄ (M) 260 122 9 50.4 46.9 AGP (M) 265 103 10 42.6 38.9 Total 785 397 25 53.8 50.6 May transmission** 610 296 9 50.3 48.5

* Sugar is 3% Sucrose (S), versus 9% Maltose (M) ** Later transmission May was only from MMS4 media, where there were another 650 to 750 smaller ELSs that were not transferred.

As can be seen from the results in Table 5, the presence of a filter paper pad leads to increased embryogenesis and green plant production as compared to the induction of embryos in liquid medium dripped on a solidified medium. Sucrose in the regeneration medium was effective in producing a high percentage of green plants, and maltose was also found to be effective. Without wishing to be bound by theory, we may conclude that this may be due to the faster release of the energy from sucrose needed for regeneration. The addition of AGP to the regeneration medium reduced the number of green plants produced. This result may indicate that AGP is important in the induction medium only at the initiation stage, but not at later stages.

Example 6

The effect of AGP on embryogenesis and plant regeneration was investigated in wheat microspores that were isolated from the genotype of Pavon 79 according to the method of the invention. Embryogenesis results are presented in Table 6 and plant regeneration values are presented in Table 7. AGP concentrations of 0, 1.5 and 10 mg / L were examined.

·· ·· ··

8 · · · · · · ·

9

Table 6. Response of isolated wheat microspore cultures to various concentrations of AGP in MMS4 medium, with and without seedlings. Two Reps cv Pavon 79.

Adjustments Total number of microspores after 10 days (D 10) number of live and divide after 10 days (d 10) number of living and non-dividing after 10 days (d 10) number dead mikrospor after 10 days (d 10) number of multicells after 10 days (d 10) number cracked vícebu- some after 10 days (d 10) number of embryos after 30 days AGP ml / l seedlings +/- presence- nost seedlings absent- nost seedlings 0 - 102805 6930 5775 83500 6600 0 0 + 105925 16305 4950 68345 12850 1125 1650 1 - 89825 22125 5650 47558 13750 400 0 + 92575 22605 2805 47175 18500 1072 2130 5 - 98015 26152 8275 34500 28345 742 0 + 102280 18272 7600 30250 39078 7080 3393 10 - 110745 33275 6250 24650 41950 4620 50 + 108305 8500 0 23550 68000 15452 4250

Total quantities on day 10 were determined by adding up from the square area and multiplying by 1.65x10 ³ . For each dish, values from three areas were read and averaged, then two replicates were averaged. To determine the number of embryos at day 30, one quarter of the dish was separated and all embryo sizes were counted.

To determine regeneration values (see Table 7), 250 embryos from one dish were transferred to the regeneration medium. A significant improvement in embryogenesis, regeneration, and% of green plants produced was the result of the addition of 10mg / L AGP, using the Pavon 79 genotype. In addition, the values illustrate that the presence of testes in mixed culture improved embryogenesis and plant regeneration. In the absence of anthrax mixed culture, significant embryogenesis was only observed at an AGP concentration of 10mg / L. Figure 5 (a) illustrates the response of wheat microspores in the presence or absence of wheat testicles and at a concentration of 0 or 10 mg / L AGP. Figure 6 illustrates the correct structure of wheat embryo developed after 4 weeks in testicle and AGP containing medium.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

9 · · «» · ····

Table 7: Regeneration of wheat microspore plants in the presence of AGP in MMS4 induction medium, cv Pavon 79.

Treatment with testicles and AGP total number mikrospor (x1000) total number embryos the number of embryos transferred to the regeneration medium number of plants % green plant % regeneration- here mg / l AGP green plants albino plants total 0 103 1650 250 37 78 115 32 46 1 90 2130 250 48 70 118 41 47 5 98 3393 250 50 70 120 42 48 10 111 4250 250 129 60 189 68 76

Example 7

Preliminary experiments on both filter paper and test fluid liquid media were compared to embryo response in induction media containing 0.5 and 10mg / L AGP. Table 8 shows the effect of AGP on an isolated culture of Pavon 79 microspores in liquid medium over a culture grown on filter paper. The results are similar for both liquid medium and filter paper at 3 concentrations, with 10 mg / l AGP showing the best response.

Table 8: Effect of AGP on an isolated culture of Pavon 79 spring wheat microspores grown in liquid medium containing MMS4 medium versus culture on filter paper on solid medium, both in a mixed culture with testicles.

AGP conc. mg / l number of cultured microspores x 1000 total number of embryos per dish 0 liquid 106 1650 0 filter cca 100 1769 1 liquid 93 2130 5 liquid 102 3393 5 filter cca 100 3080 10 liquid 108 4250 10 filter cca 100 4528

Example 8

The effect of AGP on microspore embryogenesis in the presence of a mixed testicle culture using the Chris and Pavon 79 wheat genotypes was investigated. The results in Table 9 show that the overall response of Chris wheat to AGP is higher than that of Pavon 79.

• 4 · 4 · 4444 · 4 · 4444

Table 9: Effect of AGP on microspore embryo development in the Chris and Pavon 79 wheat genotypes using 0.8 ml droplets on solid MMS4 medium with 3-4 seedlings.

Chris Pavon 79 AGP conc. in mg / l the number of repetitions average number of embryos the number of repetitions average number of embryos 0 3 4007 3 2778 5 5 5618 3 4155 10 5 9836 3 4681

Example 9

Based on the data obtained from Example 8, Tables 8 and 9, it was found that the highest AGP concentration tested (10 mg / L) gave the best embryogenesis response, with higher concentrations not being tested. In the Pavon 79 experiment, concentrations of 0, 10, 25, 50, 100, 500 and 1000 mg / L were used for a microspore culture with 4 testicles around a drop of liquid microspores on the surface of the solidified medium. The results are shown in Table 10.

Table 10: Effect of AGP concentration on microspore culture in Pavon 79 spring wheat in MMS4 liquid medium (data averaged over 4 replicates)

Conc. AGP mg / l number of ph 12 days 50.den live dead dividing vícebu- something ELS total number developing se number embryos 0 64.3 62.7 12.4 23.5 2.1 38 2862 10 69.3 58.9 23.9 33.8 8.7 66.4 4493 25 66.6 69.3 13.6 38 10.3 61.9 3538 50 47.9 75 10.7 13.2 5 28.9 471 100 ALIGN! 66.4 61 17.3 32.2 2.9 52.4 3709 500 57.3 61 19.4 27.7 4,5 51.6 3267 1000 69.3 56 14.8 28.5 7 50.3 1821

From the obtained results we can say that for optimal embryogenesis concentrations of 10 or 25 mg / l AGP are ideal. However, higher concentrations can also be used. The life of the microspores at day 12 was not significantly different from the comparative value in the absence of AGP, but the total number of developing structures increased in the presence of AGP. It was further observed that 10 to 14 days in the sAGP induction medium may be a maximum time as it appears that regeneration decreases in the AGP medium over longer periods.

• · · · · ·

Example 10

The effect of AGP concentration in culture during embryogenic induction in barley microspores was investigated. On microspores cv. Igri was investigated over a wide range of concentrations, see Table 11. Liquid FHG medium with 10 ⁵ microspores and various concentrations of AGP was added to solid FHG medium. Using a microspore drop on solidified MFHG medium, structures were counted under an inverted microscope. The ratio of live and dead microspores was not counted on these dishes, but the developing microspores were much more in the 0 mg comparator compared to wheat. Therefore, the difference in barley AGP response at day 10 is not as pronounced as in wheat. However, with wheat, the results are similar at day 50, when there was an evident continuation of development and more rapid development of AGP microspores as seen in Figure 5 (b) after 4 weeks in culture.

Table 11: Effect of AGP on development of isolated cv Igri barley microspores in FHG liquid medium after 10 days

AGP conc. in mg / l division within microspore free multicellular the total number of microspores developing 0 33280 33310 65590 2 39600 33550 73150 5 43000 38230 81230 10 45280 35750 81030 25 39880 36030 75910 50 37120 39880 77000 100 ALIGN! 42900 37730 80630 500 35890 37810 73700 1000 38500 41600 80100

Example 11

The response of isolated microspores to embryogenesis and plant regeneration in spring and winter wheat genotypes was examined visually using the method of the invention containing AGP in an induction medium. All genotypes showed a response to the method of producing embryos and green plants from microspores. Table 12 shows the relative response of each genotype on a scale of 0 to 3, where 0 represents zero response, 1 represents poor response, 2 represents good response, and 3 represents excellent response. Both embryogenesis and initiation of green plants were counted. For the winter genotypes, only 3 donor plants were available from the production reactor, so the sample was small.

• 0 · 0 0

different genotypes (0 to 3 response)

Genotypes tested response embryogenesis regeneration of green plants spring wheat Chris 3 2-3 Pavon 70 3 2-3 Pavon 76 3 2-3 Bob White S 3 3 Quantum 3 1-2 Hartog 3 1-2 winter wheat 2540 3 2 OAC Montrose 3 3 Harus 3 2 Freedom 3 2 Hanover 3 2 Fundulea 2 not analyzed AC Morley 3 2-3

The induction response in wheat is genotype-independent as well as in barley, while the regeneration response is slightly different. The results with winter wheat lines are based on only 3 donor plants that we obtained from the production reactor after the jarization. Regeneration of the Fundulea variety was not carried out. Both Pavon and Bob White are quite different varieties and therefore selection was cultivated. Bob White S is a boseless selection. Although induction in media without AGP is also good, embryo development is not as good as with AGP, and therefore regeneration is strongly affected by AGP.

All publications cited herein are incorporated by reference. Many modifications are possible without departing from the invention. It will be understood that the invention has been described herein in connection with certain examples and embodiments. Nevertheless, changes, modifications, or analogies that those skilled in the art can make are believed to be part of the invention. Also, the description of the invention is set forth by way of example, not by way of limitation, and therefore changes within the scope of the principles of the invention, as will be apparent to those skilled in the art, are considered to be part of the claims.

·· ··················· 44 44 · 9

LINKS

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Ziauddin, A., A. Marsollais, E. Simion, and K.J. Kasha. 1990. Improved plant regeneration from wheat anther and barley microspore culture using phenylacetic acid (PAA). Plant Cell rep. 11: 489-498.

Claims (52)

A method for producing an embryo comprising a microspore from a donor plant: the segment is incubated under pretreatment conditions to maintain a substantial portion of the microspores in the mononuclear phase of the G1 cell cycle: microspores are isolated from the segment: and the isolated microspores are incubated in induction media containing a hydroxyproline rich glycoprotein with high galactose side chain (GaRGP) hydrocarbon chains, thereby inducing embryogenesis and thereby producing embryos. The method of claim 1, wherein the donor plant is a cereal and the induction medium may comprise an arabinogalactan protein or a hydroxyproline rich glycoprotein. The method of claim 2, wherein the cereal is wheat or barley. The method of claim 2, wherein the arabinogalactan protein or GaRGP is present in the induction medium at a concentration of from 1 mg / L to 500 mg / L of the induction medium. The method of claim 4, wherein the arabinogalactan protein or GaRGP is present in the induction medium at a concentration of 10 mg / L to 25 mg / L induction medium. The method of claim 5, wherein the arabinogalactan protein or GaRGP is present in the induction medium for about two weeks. The method of claim 1, wherein a substantial fraction of the microspores in the mononuclear phase of the G1 cell cycle is comprised between 50% and 100%. Method according to claim 1, characterized in that the pretreatment conditions consist of a temperature of from 3 ° C to 10 ° C for 3 to 10 days and an incubation at 99 9999. 9 » 9 9 99 9 9 9 ·· · 99 · 9 · 9 99 99 9 · · 9 99 99 99 999 9 9 • 99 9 · 99 9 99 99 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 The method of claim 8, wherein the sugar alcohol is selected from the group consisting of mannitol, maltitol, sorbitol, xylitol, and any combination thereof. The method of claim 1, wherein the pretreatment conditions comprise incubation in water at a temperature of 3 ° C to 10 ° C for 7 to 28 days. The method of claim 1, wherein the plant segment comprising microspores is selected from the group consisting of shoots, flowers, ears, anthers, laths and fibers. The method of claim 1, wherein the isolated microspores are incubated in the induction medium for 3 to 14 days. The method of claim 1, wherein the induction medium comprises auxin. The method of claim 13, wherein the auxin is phenylacetic acid. The method of claim 1, wherein the induction medium comprises glutamine at a concentration of from 500 to 1,000 mg / L. 16. The method of claim 1, wherein the induction medium additionally comprises a mixed testicle culture. 17. The method of claim 16, wherein the microspore is obtained from wheat. · 9 9 9 9 9 9 9 9 9 9 9 9 9 9 99 9999 18. A method of regenerating plants from microspores, comprising collecting a plant segment comprising microspores from a donor plant; the segment is incubated under pretreatment conditions to maintain a substantial fraction of the microspores in the mononuclear phase of the G1 cell cycle; microspores are then isolated from the segment; isolated microspores are incubated in induction medium containing auxin and arabinogalactan protein in an amount of from 1 mg / l to 500 mg / l to induce embryo production; further, the embryos are incubated in differentiation medium to produce differentiated embryos; and plants are then regenerated from the differentiated embryos. 19. A plant regeneration method according to claim 18 wherein the embryos formed after incubation of the microspores are placed on a support. The method of claim 19, wherein the pad comprises filter paper. 21. The method of claim 18 wherein isolating the microspores from the segment comprises stirring or swirling the segment in an aqueous solution of 0.2 mol / L to 1.0 mol / L sugar alcohol. 22. A method of producing a cereal microspore culture, comprising incubating a microspore segment of a cereal in a medium comprising an arabinogalactan protein in an amount of from 1 mg / L to 100 mg / L for embryo formation; and the cereal is regenerated from the embryos. 23. A method of introducing a particular gene into a microspore, comprising introducing a genetic construct containing the gene into a microspore obtained by pretreatment and isolation as defined in claim 1. 24. The method of claim 23, wherein the introduction comprises particle bombardment. ♦ · φ · • · • · • · • · • · · • · • · · · · · · · · · · · · · ΦΦ φφ φφφφ 25. The method of claim 23, wherein the introduction comprises Agrobacterium-mediated transformation. 26. An embryo production method comprising collecting a plant segment containing microspores from a donor cereal: the segment is incubated under pretreatment conditions to maintain a substantial portion of the microspores in the mononuclear phase of the G1 cell cycle; microspores are isolated from the segment; and isolated microspores are incubated in induction medium containing arabinogalactan protein, thereby inducing embryogenesis and thereby producing embryos. The method of claim 26, wherein the cereal is wheat or barley. The method of claim 26, wherein the arabinogalactan protein is present in the induction medium at a concentration of from 1 mg / L to 500 mg / L of the induction medium. The method of claim 28, wherein the arabinogalactan protein is present in the induction medium at a concentration of 10 mg / L to 25 mg / L induction medium. 30. The method of claim 29, wherein the arabinogalactan protein is present in the induction medium for about two weeks. 31. The method of claim 26 wherein a substantial fraction of the microspores in the mononuclear phase of the G1 cell cycle are comprised between 50% and 100% during incubation. A method according to claim 26, wherein the pretreatment conditions comprise a temperature of from 3 ° C to 10 ° C for 3 to 10 days and incubation under the pretreatment conditions is carried out in an aqueous solution containing from 0.2 mol / l to 1 ° C. , 0 mol / l sugar alcohol. »Toto toto to to to to to ► ► ► ► ► ► ► * * * * * * * * * * * * * * * * et et · ··· · 33. The method of claim 32, wherein the sugar alcohol is selected from the group consisting of mannitol, maltitol, sorbitol, xylitol, and any combination thereof. The method of claim 26, wherein the pretreatment conditions include incubation in water at a temperature of 3 ° C to 10 ° C for 7 to 28 days. 35. The method of claim 26, wherein the plant segment comprising microspores is selected from the group consisting of shoots, flowers, ears, anthers, laths and fibers. 36. The method of claim 26, wherein the isolated microspores are incubated in the induction medium for a period of from 3 to 14 days. 37. The method of claim 26, wherein the induction medium comprises auxin. 38. The method of claim 37, wherein the auxin is phenylacetic acid. The method of claim 26, wherein the induction medium comprises glutamine at a concentration of 500 to 1,000 mg / L. 40. The method of claim 26, wherein the induction medium additionally comprises a mixed testicle culture. 41. The method of claim 40, wherein the microspore is harvested from wheat. A microspore obtained from an induction medium as defined in claim 26 prior to embryogenesis. The embryo produced from the microspore of claim 42. 9 9 9 9 499 499 9 9 38 · * ·· · ϊ i * 499 9 9 9 * 999 49 9 4 · 9 «· * 9994 44. The plant produced from the embryo of claim 43. 45. A seed derived from a plant according to claim 44. 46. A method of producing a microspore composition comprising collecting a plant segment comprising microspores from a donor plant; the segment is incubated under pretreatment conditions to maintain a substantial fraction of the microspores in the mononuclear phase of the cell cycle; microspores are isolated from the segment; isolated microspores are incubated in an induction medium containing arabinogalactan protein to produce a microspore composition that, after 10 days of incubation, contains more than 25% viable microspores. 47. A microspore composition comprising after 10 days incubation more than 25% viable microspores. 48. A method of producing a microspore composition comprising collecting a plant segment comprising microspores from a donor plant; the segment is incubated under pretreatment conditions to maintain a substantial fraction of the microspores in the mononuclear phase of the cell cycle; microspores are isolated from the segment; isolated microspores are incubated in an induction medium containing arabinogalactan protein to produce a microspore composition that, after 10 days of incubation, contains more than 15% of multicellular microspores. 49. A microspore composition comprising after 10 days of incubation more than 15% of multicellular microspores. A microspore obtained from an induction medium as defined in claim 1 prior to embryogenesis. An embryo produced from the microspore of claim 50. The plant produced from the embryo of claim 51.